Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis

The primary quinone acceptor radical anion Q(A)· (a menaquinone-9) is studied in reaction centers (RCs) of Rhodopseudomonas viridis in which the high-spin non-heme Fe is replaced by diamagnetic Zn. The procedure for the iron substitution, which follows the work of Debus et al. [Debus, R. J., Feher, G., and Okamura, M. Y. (1986) Biochemistry 25, 2276-2287], is described. In Rps. viridis an exchange rate of the iron of ~50% ± 10% is achieved. Time-resolved optical spectroscopy shows that the ZnRCs are fully competent in charge separation and that the charge recombination times are similar to those of native RCs. The g tensor of Q(A)· in the ZnRCs is determined by a simulation of the EPR at 34 GHz yielding g(x) = 2.00597 (5), g(y) = 2.00492 (5), and g(z) = 2.00216 (5). Comparison with a menaquinone anion radical (MQ·) dissolved in 2-propanol identifies Q(A)· as a naphthoquinone and shows that only one tensor component (g(x)) is predominantly changed in the RC. This is attributed to interaction with the protein environment. Electron-nuclear double resonance (ENDOR) experiments at 9 GHz reveal a shift of the spin density distribution of Q(A)· in the RC as compared with MQ· in alcoholic solution. This is ascribed to an asymmetry of the Q(A) binding site. Furthermore, a hyperfine coupling constant from an exchangeable proton is deduced and assigned to a proton in a hydrogen bond between the quinone oxygen and surrounding amino acid residues. By electron spin-echo envelope modulation (ESEEM) techniques performed on Q(A)· in the ZnRCs, two N nuclear quadrupole tensors are determined that arise from the surrounding amino acids. One nitrogen coupling is assigned to a N(δ(1))-H of a histidine and the other to a polypeptide backbone N-H by comparison with the nuclear quadrupole couplings of respective model systems. Inspection of the X-ray structure of Rps. viridis RCs shows that His(M217) and Ala(M258) are likely candidates for the respective amino acids. The quinone should therefore be bound by two H bonds to the protein that could, however, be of different strength. An asymmetric H-bond situation has also been found for Q(A)· in the RC of Rhodobacter sphaeroides. Time-resolved electron paramagnetic resonance (EPR) experiments are performed on the radical pair state P·Q(A)· in ZnRCs of Rps. viridis that were treated with o-phenanthroline to block electron transfer to Q(B). The orientations of the two radicals in the radical pair obtained from transient EPR and their distance deduced from pulsed EPR (out-of-phase ESEEM) are very similar to the geometry observed for the ground state PQ(A) in the X-ray structure [Lancaster, R., Michel, H. (1997) Structure 5, 1339]

Structure of the metal-water complex in Ras x GDP studied by high-field EPR spectroscopy and ³¹P NMR spectroscopy

The small GTPase Ras plays a key role as a molecular switch in the intercellular signal transduction. On Mg(2+) --> Mn(2+) substituted samples, the first ligand sphere of the metal ion in the inactive, GDP-bound Ras has been studied by continuous wave EPR at 94 GHz (W-band). Via replacement of normal water with (17)O-enriched water, the (17)O--(55)Mn superhyperfine coupling was used to determine the number of water ligands bound to the metal ion. In contrast to EPR data on frozen solutions and X-ray data from single crystals where four direct ligands to the metal ion are found, the wild-type protein has only three water ligands bound in solution at room temperature. The same number of water ligands is found for the mutant Ras(T35S). However, for the alanine mutant in position 35 Ras(T35A) as well as for the oncogenic mutant Ras(G12V), four water ligands can be observed in liquid solution. The EPR studies were supplemented by (31)P NMR studies on the Mg(2+) x GDP complexes of the wild-type protein and the three mutants. Ras(T35A) exists in two conformational states (1 and 2) with an equilibrium constant K(1)(1,2) of approximately 0.49 and rate constants k(1--1) which are much smaller than 40 s(-1) at 298 K. For wild-type Ras and Ras(T35S), the two states can also be observed with equilibrium constants K(1)(1,2) of approximately 0.31 and 0.21, respectively. In Ras(G12V), only one conformational state could be detected

A High-Field EPR Study of P700+• in Wild-Type and Mutant Photosystem I from Chlamydomonas reinhardtii

High-frequency, high-field EPR at 330 GHz was used to study the photo-oxidized primary donor of photosystem I (P700(+.)) in wild-type and mutant forms of photosystem I in the green alga Chlamydomonas reinhardtii. The main focus was the substitution of the axial ligand of the chlorophyll a and chlorophyll a' molecules that form the P-700 heterodimer. Specifically, we examined PsaA-H676Q, in which the histidine axial ligand of the A-side chlorophyll a' (P-A) is replaced with glutamine, and PsaB-H656Q, with a similar replacement of the axial ligand of the B-side chlorophyll a (P-B), as well as the double mutant (PsaA-H676Q/PsaB-H656Q), in which both axial ligands were replaced. We also examined the PsaA-T739A mutant, which replaces a threonine residue hydrogen-bonded to the 13(1)-keto group Of PA with an alanine residue. The principal g-tensor components of the P700(+.) radical determined in these mutants and in wild-type photosystem I were compared with each other, with the monomeric chlorophyll cation radical (Chl(z)(+.)) in photosystem II, and with recent theoretical calculations for different model structures of the chlorophyll a(+) cation radical. In mutants with a modified P-B axial ligand, the g(zz) component of P-700(+.) was shifted down by up to 2 x 10(-4), while mutations near P-A had no significant effect. We discuss the shift of the gzz component in terms of a model with a highly asymmetric distribution of unpaired electron spin in the P-700(+.) radical cation, mostly localized on PB, and a deviation of the PB chlorophyll structure from planarity due to the axial ligand